Development and validation of a new fluorescence-based bioassay for aquatic macrophyte species

Bioassays with unicellular algae are frequently used as ecotoxicological test systems to evaluate the toxicity of contaminated environmental samples or chemicals. In contrast, aquatic macrophyte test systems are still rarely used as they are laborious to handle because species exhibit distinct ecological requirements. The aim of this study was to establish a fast and reproducible measuring system for aquatic macrophyte species to overcome those limitations for use. Thus, a newly developed pulse-amplitude modulated chlorophyll fluorometer (Imaging-PAM) was applied as an effect detection in short-term bioassays with aquatic macrophyte species. This multiwell-plate-based measuring device enables the incubation and measurement of up to 24 samples in parallel. The Imaging-PAM was used (i) to establish and validate the sensitivity of the test systems to three Photosystem II (PSII) inhibitors (atrazine, prometryn, isoproturon), (ii) to compare the test systems with established biotests for macrophytes and (iii) to define necessary time scales in aquatic macrophyte testing. The results showed that fluorescence-based measurements with the Imaging-PAM allow rapid and parallel analysis of large amounts of aquatic macrophyte samples and of toxicants effects of the PSII inhibitors tested on aquatic macrophytes. Measurements revealed a good correlation between obtained median effective concentrations (EC50s) for the new and the established biotest systems. Hence, the Imaging-PAM measuring device is a promising tool to allow fast chemical effect screening for high amounts of samples with little time and material and thus offers scope for high-throughput biotesting using aquatic macrophyte species.     

AMEG: the new SETAC advisory group on aquatic macrophyte ecotoxicology

Introduction and background

Primary producers play critical structural and functional roles in aquatic ecosystems; therefore, it is imperative that the potential risks of toxicants to aquatic plants are adequately assessed in the risk assessment of chemicals. The standard required macrophyte test species is the floating (non-sediment-rooted) duckweed Lemna spp. This macrophyte species might not be representative of all floating, rooted, emergent, and submerged macrophyte species because of differences in the duration and mode of exposure; sensitivity to the specific toxic mode of action of the chemical; and species-specific traits (e.g., duckweed's very short generation time).

Discussion and perspectives

These topics were addressed during the workshop entitled “Aquatic Macrophyte Risk Assessment for Pesticides” (AMRAP) where a risk assessment scheme for aquatic macrophytes was proposed. Four working groups evolved from this workshop and were charged with the task of developing Tier 1 and higher-tier aquatic macrophyte risk assessment procedures. Subsequently, a SETAC Advisory Group, the Macrophyte Ecotoxicology Group (AMEG) was formed as an umbrella organization for various macrophyte working groups. The purpose of AMEG is to provide scientifically based guidance in all aspects of aquatic macrophyte testing in the laboratory and field, including prospective as well as retrospective risk assessments for chemicals. As AMEG expands, it will begin to address new topics including bioremediation and sustainable management of aquatic macrophytes in the context of ecosystem services.     

漂浮植物塘处理农村分散生活污水的应用

通过现场实验评价了漂浮植物塘配合化粪池处理农村分散生活污水的效果。实验结果表明,漂浮植物有效地抑制了污染水体中藻类的生长;在平均水力停留时间为36 d,COD、TN、TP平均污染负荷分别为3.1、0.86和0.056 g/(m2.d)的条件下,大薸塘对COD、TN、TP的平均去除率分别为68.5%、89.9%和85.2%,出水COD、TN和TP平均浓度分别为47、4.15和0.40 mg/L,达到GB18918-2002中的一级A标准。在水乡地区利用漂浮植物与农村宅河构造漂浮植物处理系统,是一种深度处理化粪池出水、控制农村生活污水对河道造成污染的有效措施。     

Aquatic arsenic: phytoremediation using floating macrophytes

Phytoremediation, a plant based green technology, has received increasing attention after the discovery of hyperaccumulating plants which are able to accumulate, translocate, and concentrate high amount of certain toxic elements in their above-ground/harvestable parts. Phytoremediation includes several processes namely, phytoextraction, phytodegradation, rhizofiltration, phytostabilization and phytovolatilization. Both terrestrial and aquatic plants have been tested to remediate contaminated soils and waters, respectively. A number of aquatic plant species have been investigated for the remediation of toxic contaminants such as As, Zn, Cd, Cu, Pb, Cr, Hg, etc. Arsenic, one of the deadly toxic elements, is widely distributed in the aquatic systems as a result of mineral dissolution from volcanic or sedimentary rocks as well as from the dilution of geothermal waters. In addition, the agricultural and industrial effluent discharges are also considered for arsenic contamination in natural waters. Some aquatic plants have been reported to accumulate high level of arsenic from contaminated water. Water hyacinth (Eichhornia crassipes), duckweeds (Lemna gibba, Lemna minor, Spirodela polyrhiza), water spinach (Ipomoea aquatica), water ferns (Azolla caroliniana, Azolla filiculoides, and Azolla pinnata), water cabbage (Pistia stratiotes), hydrilla (Hydrilla verticillata) and watercress (Lepidium sativum) have been studied to investigate their arsenic uptake ability and mechanisms, and to evaluate their potential in phytoremediation technology. It has been suggested that the aquatic macrophytes would be potential for arsenic phytoremediation, and this paper reviews up to date knowledge on arsenic phytoremediation by common aquatic macrophytes.     

Defining reference conditions for acidified waters using a modern analogue approach

Analogue matching is a palaeolimnological technique that aims to find matches for fossil sediment samples from a set of modern surface sediment samples. Modern analogues were identified that closely matched the pre-disturbance conditions of eight of the UK Acid Waters Monitoring Network (AWMN) lakes using diatom- and cladoceran-based analogue matching. These analogue sites were assessed in terms of hydrochemistry, aquatic macrophytes and macro-invertebrates as to their suitability for defining wider hydrochemical and biological reference conditions for acidified sites within the AWMN. The analogues identified for individual AWMN sites show a close degree of similarity in terms of their hydrochemical characteristics, aquatic macrophytes and, to a lesser extent, macro-invertebrate fauna. The reference conditions of acidified AWMN sites are inferred to be less acidic than today and to support a wider range of acid-sensitive aquatic macrophyte and macro-invertebrate taxa than that recorded in the AWMN lakes over the period of monitoring since 1988.     

Effects of planting system design on the toxicological sensitivity of Myriophyllum spicatum and Elodea canadensis to atrazine

The triazine herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-trazine) was selected as a chemical stressor in an investigation of how toxicological responses of individually grown macrophytes reflect those of plants grown in more natural model populations and two-species communities. Phytotoxicity of the compound to Myriophyllumspicatum L. and Elodeacanadensis Michx. was assessed under semi-natural field conditions using 12000l outdoor microcosms. Exposure concentrations of 25, 50, 100, 250mugl(-1) plus controls (n=3) were evaluated, selected to fall within a range of concentrations known to produce a toxic response in the tested macrophytes, and effective concentrations required to cause a decrease in biomass endpoints by 10%, 25%, and 50% were estimated. The sensitivities of aquatic plants to atrazine did not differ substantially between planting systems, and few interactions between the effects of the planting method and atrazine effects on macrophyte biomass were detected using a two-way ANOVA. A lack of significant differences in biomass and relative growth rate measures between plants grown under the various test systems also indicated that interactions between and among species did not influence growth of plants in the model population and communities. Under these test conditions, the use of the "cone-tainer" method provided estimates of toxicity consistent with those from plants grown in assemblages, and potential interactions between plants were not found to modify the response of macrophytes to atrazine.     

Relating cadmium concentrations in three macrophyte-associated freshwater invertebrates to those in macrophytes,water and sediments

We found that Cd concentrations in three species of macrophyte-associated invertebrates (the gastropods Bithynia tentaculata and Physa gyrina, and the amphipod Gammarus fasciatus) collected at twenty sites along the St. Lawrence River were correlated with Cd concentrations in their main food source, i.e. macrophytes and associated periphyton. Cd in these invertebrates was not significantly correlated with Cd concentrations in the sediments (even when corrections for iron oxide or organic carbon content were applied) or to calculated free-cadmium concentrations in the water. Cd levels in juveniles of B. tentaculata were very tightly linked to Cd concentrations in macrophytes, which is consistent with the close relationship that these organisms have developed with macrophytes. Linear models predicting Cd levels in juveniles of B. tentaculata from Cd levels in different macrophyte species were strong (R2 = 0.69-0.90). Analysis of covariance on these models showed no statistical difference of slope or intercept for any of the macrophyte species, except Vallisneria americana. We suggest that the macrophyte-periphyton complex is a key link in the transfer of cadmium to some aquatic invertebrates in the littoral zone of the St. Lawrence River and that macrophytes and their associated epiphytes should also be used as biomonitors.     

Hydroponic uptake of atrazine and lambda-cyhalothrin in Juncus effusus and Ludwigia peploides

Phytoremediation encompasses an array of plant-associated processes known to mitigate contaminants from soil, sediment, and water. Modification of pesticides associated with agricultural runoff includes processes directly associated with aquatic macrophytes in addition to changes in soil geochemistry and associated rhizospheric degradation. Remediation attributes of two vegetative species common to agricultural drainages in the Mississippi Delta, USA, were assessed using atrazine and lambda-cyhalothrin. Concentrations used in 8-d hydroponic exposures were calculated using recommended field applications and a 5% runoff model from a 0.65-cm rainfall event on a 2.02-ha field. While greater atrazine uptake was measured in Juncus effusus, greater lambda-cyhalothrin uptake occurred in Ludwigia peploides. Maximum pesticide uptake was reached within 48h for each exposure and subsequent translocation of pesticides to upper plant biomass occurred in macrophytes exposed to atrazine. Sequestration of 98.2% of lambda-cyhalothrin in roots of L. peploides was measured after 8d. Translocation of lambda-cyhalothrin in J. effusus resulted in 25.4% of pesticide uptake partitioned to upper plant biomass. These individual macrophyte remediation studies measured species- and pesticide-specific uptake rates, indicating that seasonality of pesticide applications and macrophyte emergence might interact strongly to enhance mitigation capabilities in edge-of-field conveyance structures.     

Phytoremediation Potential of Aquatic Macrophyte, <Emphasis Type="Italic">Azolla</Emphasis>

Aquatic macrophytes play an important role in the structural and functional aspects of aquatic ecosystems by altering water movement regimes, providing shelter to fish and aquatic invertebrates, serving as a food source, and altering water quality by regulating oxygen balance, nutrient cycles, and accumulating heavy metals. The ability to hyperaccumulate heavy metals makes them interesting research candidates, especially for the treatment of industrial effluents and sewage waste water. The use of aquatic macrophytes, such as Azolla with hyper accumulating ability is known to be an environmentally friendly option to restore polluted aquatic resources. The present review highlights the phytoaccumulation potential of macrophytes with emphasis on utilization of Azolla as a promising candidate for phytoremediation. The impact of uptake of heavy metals on morphology and metabolic processes of Azolla has also been discussed for a better understanding and utilization of this symbiotic association in the field of phytoremediation.     

15N Metabolic test for the determination of phytotoxic effects of chemicals and contaminated environmental samples

A stable isotope¹⁵N-nitrogen test (ESIMA = Ecotoxicological Stable Isotope Metabolic Assay) was developed to assess biological effects and the potential toxicological hazard of chemicals and contaminated environmental samples on plant metabolism. The assay measures the effect of toxicants on the incorporation of a¹⁵N labelled tracer into the total nitrogen fraction (both the nonprotein and protein fraction) of plants. Segments ofPisum arvense epicotyls are used as test substrates because of their high metabolic activity. The plant material is incubated under standardised conditions for two hours; subsequently¹⁵N incorporation is analysed by determining the¹⁵N abundance (¹⁵N atom-%) in the epicotyl segments. The effects of toxicants are evaluated by comparing the¹⁵N incorporation rates of control tissue and epicotyl segments exposed to individual chemicals or complex environmental samples. The specificity and sensitivity of effects as indicated by ESIMA were compared with effects as measured by two established ecotoxicological bioassays, the pollen tube growth test using pollen ofNicotiana sylvestris and the bacterial luminescence inhibition test using pollen ofPhotobacterium phosphoreum. The results of the study clearly indicate the suitability of ESIMA for assessing toxic impacts on plant nitrogen metabolism. Prof. Dr. habil. Hans Faust dedicated to his 70^th birthday.     

Sensitivity of Bidens laevis L. to mutagenic compounds. Use of chromosomal aberrations as biomarkers of genotoxicity

The wetland macrophyte Bidens laevis possesses suitable cytological characteristics for genotoxicity testing. To test its sensitivity as compared to terrestrial plants species currently in use in standardized assays, Methyl Methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU) and Maleic Hydrazide (HM) were used. On the other hand, the insecticide Endosulfan (ES) - an environmentally relevant contaminant - was assayed in seeds and two-month old plants. Mitotic Index (MI), frequency of Chromosome Aberrations in Anaphase-Telophase (CAAT) and frequency of Abnormal Metaphases (AM) were analyzed. MH, MMS and ENU caused a significant decrease of the MI. MMS was aneugenic whereas MH and ENU were both aneugenic and clastogenic. ES caused a significant concentration-dependent increase of total- and aneugenic-CAAT in roots and a significant high frequency of AM at high concentrations. Because of its sensitivity to mutagenic substances, B. laevis can be regarded as a reliable and convenient species for genotoxicity assays especially if aquatic contaminants are evaluated.     

The impact of pesticides toward parrotfeather when applied at the predicted environmental concentration

Pesticides pose a serious risk for aquatic macrophytes in the environment. They are also detrimental to the rooted macrophytes used in bioassays for assessment. Currently, no data is available for impact of pesticides toward parrotfeather when present at the predicted environmental concentration. The calculated expected environmental concentration was applied to the plants and the effect was compared. Eight of the 18 pesticides showed significantly different impact. All of the other tested pesticides induced a significant change in pigment content of parrotfeather. The RQ values for risk quotient had a value higher than 0.5, so need regulatory action for environment. This study may be the first to evaluate the predicted environmental concentrations reported by pesticide registration in Europe. Additional studies are required to test all pesticides within one group since the compounds tested may depict a wide toxicity level. Furthermore, the tests should include more than one macrophyte, e.g. one rooted and one non-rooted species, in order to provide a better understanding on pesticide toxicity.     

Phytotoxicity of diuron alone and in combination with copper or folpet on duckweed (Lemna minor)

The photosystem II-herbicide diuron is widely used for weed control in Champagne's vineyards. Its important use and its relative persistence make it of particular interest for ecotoxicological studies. Toxicity of diuron was assessed on Lemna minor L., a representative aquatic macrophyte regularly used for toxicological studies. Toxicity assessments were based on inhibition of growth and total chlorophyll content of L. minor cultures after 7 days. Growth was inhibited and IC(50) and IC(90) were, respectively, 25 and 60 microg l(-1), but chlorophyll content of L. minor increased in response to the herbicide. When diuron was combined with copper, growth inhibition of L. minor depended on the concentrations of both chemicals. For some concentrations, combination of these chemicals resulted in a slight (but non-significant) antagonism. Additivity was observed for all other mixtures. When diuron was combined with folpet, growth and chlorophyll content of L. minor only depended on the concentration of the herbicide. Diuron was also found to prevent the copper-induced decrease of chlorophyll content when it was combined with this metal. A multifactorial model was found more appropriate to characterize interactions between pesticides than Abott's model.     

夏季富营养化滆湖中沉水植物群落重建及水质净化效果

通过滆湖原位围隔试验分析了不同单种及混种沉水植物群落随环境变化的生长状况及对水质的净化效果.结果表明:(1)夏季,沉水植物生物量的平均值与透明度/水位存在显著线性相关性,进一步表明沉水植物的生长在很大程度上依赖于光强和水深.(2)各植物区(除马来眼子菜区外)的高锰酸盐指数、TN高于空白对照区.(3)对比空白对照区,苦草...     

Comparing growth development of <Emphasis Type="Italic">Myriophyllum</Emphasis> spp. in laboratory and field experiments for ecotoxicological testing

BACKGROUND, GOALS AND SCOPE: Risk assessment of herbicides and the evaluation of contaminated sediments based on algae and the macrophyte Lemna sp. alone may underestimate the potential hazard of certain compounds. Therefore, various test systems with Myriophyllum spp. have been developed recently to assess the phytotoxicity in surface waters and natural sediments. In the present study, experiments investigating the growth development of Myriophyllum spp. were performed in the laboratory under defined conditions and in mesocosms under environmentally realistic exposure conditions to evaluate the suitability of these species as potential standard test organisms in ecotoxicological testing. This study provides data on the endpoints biomass, plant length and root development. MATERIALS AND METHODS: Six independent experiments were performed to investigate the plant development of Myriophyllum spp. under control conditions. The main difference in the experiments was the complexity of the test systems ranging from simple laboratory experiments to complex outdoor mesocosm studies. At the start of each experiment, uniform cuttings of Myriophyllum spp. were placed in vessels with or without sediments to reduce variability between replicates. The endpoints considered in this investigation were biomass (fresh weight of the whole plant), length of the main shoot, length of the side shoots, total length of the plant (calculated from the length of the main and side shoots) and root formation. Root to shoot ratios were calculated as a further measure for plant development. Relative growth rates (RGR) based on plant length (RG(L)R) and on biomass (RG(B)R) were calculated. RESULTS: Despite the various experimental conditions, comparable growth was obtained in all test systems and the variability of endpoints, such as total length and biomass of plants, was low. It was observed that the RGR of M. spicatum in the simple laboratory test system with sediment were comparable to growth data obtained for M. verticillatum and M. spicatum grown in indoor and outdoor mesocosms, thus indicating that Myriophyllum growth tends to increase by the addition of sediment. High variability was determined for the endpoints length of the side shoots, total root length and biomass of roots. DISCUSSION: One challenge for a test design to investigate phytotoxicity on aquatic plants is to obtain good growth of the plants. From the results, it can be concluded that the experimental conditions in the various test systems were suitable to study the plant development of Myriophyllum spp. because obtained growth rates were comparable between laboratory and field investigations. Another challenge for developing a plant biotest system is the definition of sensitive endpoints. Low variability is preferred to detect minor effects of chemicals or polluted sediments on plant development. In our studies, the variability of the endpoints biomass and total length of plant was low and, therefore, they have much potential as endpoints for assessing toxicity. CONCLUSIONS: The methodologies presented in this study have applications within the risk assessment for aquatic plants and have the advantage of assessing effects taking into account the relevant exposure pathways via water and/or sediment for compounds under investigation. RECOMMENDATIONS AND PERSPECTIVES: Setting safe quality criteria for surface water and sediments is one of the challenges authorities are facing today. Myriophyllum spp. is recommended as suitable test species to investigate phytotoxicity in surface water and sediments. These results, thus, might serve as a basis for the compilation of a new harmonised guideline for ecotoxicological testing with aquatic macrophytes.     

Phytotoxicity of silver nanoparticles to Lemna minor L

The use of silver nanoparticles (AgNPs) in commercial products has increased significantly in recent years. Although there has been some attempt to determine the toxic effects of AgNPs, there is little information on aquatic plants which have a vital role in ecosystems. This study reports the use of Lemna minor L. clone St to investigate the phytotoxicity of AgNPs under modified OECD test conditions. AgNPs were synthesised, characterised and subsequently presented to the L. minor. Results showed that inhibition of plant growth was evident after exposure to small (∼20 nm) and larger (∼100 nm) AgNPs at low concentrations (5 μg L⁻¹) and this effect became more acute with a longer exposure time. There was a linear dose-response relationship after 14 d exposure. Using predicted environmental concentrations for wastewaters it was found that AgNPs may pose a significant potential risk to the environment.     

Growth assays with mixed cultures of cyanobacteria and algae assessed by in vivo fluorescence: One step closer to real ecosystems?

A growth toxicity assay with mixed cultures of cyanobacteria and algae using in vivo fluorescence is presented. Test organisms (the green alga Pseudokirchneriella subcapitata and the cyanobacterium Aphanothece clathrata) growing alone and in a mixture were exposed to selected chemicals. P. subcapitata featured a higher sensitivity to toxicants in the presence of A. clathrata compared to the single species assay. On the other hand, growth of a cyanobacterium was not affected by the presence or absence of the green alga. The proposed method seems to be suitable for pre-screening studies of toxicants (algistatic agents, herbicides) applied into the aquatic environment and for the assessment of their impact on natural phytoplankton communities.     

Diquat associated with copper sources for algae control: Efficacy and ecotoxicology

The aims of this research were to evaluate the efficacy of copper oxychloride (CuCl₂.3Cu(OH)₂), copper hydroxide (Cu(OH)₂) and diquat (1.1′-ethylene-2.2′-bipyridyldiylium dibromide), isolated and in association with 0.1% of both copper sources, in the control of the unicellular algae Ankistrodesmus gracilis and the filamentous algae Pithophora kewesis, and to determine the acute toxicity of the tested chemicals in Hyphressobrycon eques, Pomacea canaliculata, Lemna minor and Azolla caroliniana. The efficacy was estimated by the methods of chlorophyll a and pheophytin a readings, changed into growth inhibition percentage. Both algae were exposed to the following concentrations: 0.2; 0.4; 0.8; 1.2 mg L^?1 of diquat and its association with the copper sources; and 0.1; 0.3; 0.5; 0.7; 1.0 and 1.5 mg L^?1 in the isolated applications of copper hydroxide and copper oxychloride. An untreated control was kept. The acute toxicity was estimatedby 50% lethal concentration (LC50). The copper sources were effective for A. gracilis control, at rates as high as 0.1 mg L^?1 (>95% efficacy). Isolated diquat and its association with copper hydroxide were both effective at rates as high as 0.4 mg L^?1, with 95 and 88% control efficacy, respectively. The copper oxychloride was effective at 0.2 mg L^?1, with 93% efficacy. None of the tested chemicals and associations was effective on P. kewesis control. The most sensitive non target organism to the tested chemicals was L. minor; the less sensitive was H. eques.     

Bioassays with Vibrio fischeri for the assessment of delayed toxicity

The standardized bioluminescence assay with Vibrio fischeri underestimates the aquatic toxicity of chemicals which interfere with metabolic pathways supporting long term processes like growth and reproduction due to its short incubation time (30 min). Therefore this short term assay was compared with two alternative bioassays with prolonged incubation times using the same test organism: the growth inhibition assay (7 h) and the long term bioluminescence assay (24 h). Two sets of compounds were selected to reflect acute and delayed toxicity. The first group comprised pentachlorophenol, dodecylpyridiniumbromide and 3,4-dichloroaniline and the second nalidixic acid, chloramphenicol and streptomycinsulfate. The effects of compounds with acute toxicity are determined with similar sensitivity in all bioassays. Substances with delayed toxicity show only minor or no toxicities in the standardized short term bioassay but severe effects in both long term bioassays independent of the parameter used. It is concluded that the standardized short term bioluminescence assay exhibits serious limitations for the assessment of aquatic toxicity. The long term bioassays, however, may help to overcome these limitations.